Summary:

A crucial first step in the development of a general molecular manufacturing
technology is the ability to build complex molecular devices by linking
molecular building blocks together into arbitrarily complex structures.

Such molecular devices (nanodevices) will also have many diverse and profitable
near-term applications as commercial products.

DNA-Guided Assembly of Proteins (DGAP) provides a novel approach for the
fabrication of molecular devices and promises simpler and quicker development
from current technology than other proposals.

Introduction:

A General Molecular Manufacturing Technology

Drexler (1981, 1986, 1992)
has argued that it is possible to develop a technology capable of inexpensively
fabricating large and complex structures to atomic precision, meaning with
each atom placed to contribute to the designed function of the product.
This proposed technology has been named molecular manufacturing, or molecular
nanotechnology, or simply nanotechnology. Theoretical studies (Drexler,
1992) show that such manufactured products will have properties and
functions many orders of magnitude beyond the properties and functions
of products manufactured using current technology, and will in addition
be inexpensive.

Assemblers, Replicators and Mechanosynthesis

Molecular manufacturing will require developing a class of programmable
machines capable of covalently joining atoms or molecular fragments into
any of a large number of possible bonding arrangements. Thus such machines,
which have been termed assemblers, will be able to fabricate almost any
device whose construction can be specified in atomic detail.

Assemblers that have been designed to be able to make exact copies of
themselves have been termed replicators.

A key capability of assemblers will be the ability to do mechanosynthesis.
Drexler (1992) has defined mechanosynthesis as "Chemical
synthesis controlled by mechanical systems operating with atomic-scale
precision, enabling direct positional selection of reaction sites ..."

The assemblers envisioned by Drexler for use in molecular manufacturing
would be very strong, stiff structures that would work in a vacuum, be
able to handle very reactive chemical moieties, such as free radicals,
and be able to position such species to a precision of better than 0.1
nm. It is difficult to see how such complex devices could be manufactured
directly except by already existing, more primitive assemblers.

Proto-Assemblers

It is expected (Drexler, 1992) that the ultimate assemblers
will thus be developed through several generations of devices, with very
crude and limited tools being used to make better tools, eventually culminating
in the production of the desired general purpose replicating assemblers.
Early generations of assemblers, relatively limited in their capabilities,
have been termed proto-assemblers.

The obvious bottleneck to the development of nanotechnology is the identification
of the first generation of replicating, programmable assemblers that
can be fabricated using current laboratory technology, or incremental developments
of current technology, which will not require for their fabrication already
existing replicating assemblers. To be useful, such proto-assemblers must
be able, with appropriate programming, to fabricate second generation assemblers
with enhanced synthetic capabilities.

It has been suggested (Drexler, 1992) that early
generations of assemblers will deal not with very reactive single atoms
or small groups of atoms in a vacuum, but rather with larger molecular
building blocks (MBBs) in solution. Drexler considered two approaches:
(1) Brownian assembly of fairly large MBBs, each of which is a polymer
designed to fold into a specific shape, and (2) mechanosynthetic assembly
of small MBBs guided by a scanning force microscope.

Building molecular devices (nanodevices) by Brownian assembly of engineered
proteins or other polymers, designed to fold and form matching complementary
surfaces with which they bind noncovalently to each other in specific arrangements,
presents very formidable design challenges, which seem unlikely to be solved
within the next few years at current rates of progress.

As for the second approach, initial analysis (Krummenacker,
1994) reveals that the choice of appropriate MBBs and methods of linking
them together in precise three-dimensional networks is a very difficult
problem in organic chemistry.

Therefore, there seems to not be an obvious way to make even proto-assemblers
starting with current technology.

Protein Molecular Machines as the Earliest Proto-Assemblers

Drexler has suggested (Drexler, 1981, 1992)
that the initial, crudest steps toward molecular manufacturing capability
could be taken using protein-like molecular machines, and that development
pathways exist which lead from them to more general molecular manufacturing
technologies, for example the use of small synthetic organic groups, attached
to protein-based machines, as tools for positional control of reactions.

Drexler discussed the engineering of protein-like molecules having complementary
surfaces for self-assembly, and proposed that designing proteins to fold
in a predictable fashion was much easier than predicting the folding pattern
of natural proteins. He suggested methods for increasing the stability
of the desired conformation of designed proteins. This approach can almost
certainly eventually be made to work, but it entails a formidable challenge
in protein design, as stated earlier.

DNA-Guided Assembly of Proteins (DGAP) for the Fabrication of Nanodevices

A novel approach invented by Bruce Smith uses DNA-protein conjugates produced
by biotechnology as MBBs. However, rather than designing variations of
proteins or protein-like polymers to fold into desired tertiary and quaternary
structures to be used as artificial complementary surfaces for binding,
this invention utilizes the well understood complementarity of DNA sequences
to assemble proteins into specific configurations. After being positioned
relative to one another using DNA complementarity, MBBs would be joined
by forming covalent bonds, using solution chemistry of the kind which is
familiar and routine in organic chemistry.

Drexler pointed in this direction in general terms in 1992 ("Nanosystems"
p.446, after discussing the work of Ned Seeman (1991)):

"The ability of complementary segments of DNA to bind to one
another provides a powerful, readily controllable mechanism for guiding
the Brownian assembly of molecules. Hybrid structures of DNA and other
macromolecules may prove particularly attractive."

Protein-DNA conjugates, with different DNA sequences attached at
specific points on the protein surface, thus become the MBBs to be assembled
by Brownian motion to build nanodevices. One of the advances in Smith's
proposal beyond what Drexler suggested in 1992 is that "loose" binding
of complementary DNA strands on different protein molecules confers specificity
on subsequent rigid, covalent binding between protein MBBs by holding the
MBBs accurately enough that each covalent binding site is accessible only
to its intended partner. The resulting structures will be strong enough
to use as components of molecular machines able to actively position similar
MBBs, and to perform some forms of positionally-controlled chemical synthesis.
A second advance is how to achieve a defined spatial arrangement of several
different DNA sequences on the surface of the protein, even though it is
not feasible to use a different attachment chemistry at each attachment
site on the protein.

The modifications required of the proteins for permanent bonding are
thus much simpler than if the proteins had to be redesigned to have complementary
surfaces. An additional advantage of this approach is that the library
of potential MBBs includes the vast collection of proteins produced by
biological evolution (many of which have known structures) as well as synthetic
variants thereof. Thus a wide variety of MBBs with specific functions (structural,
ligand-binding, and catalytic) can be included in these first generation
nanodevices.

The DGAP Invention:

Motivation for DGAP

The hardest problem in getting from today's technology to primitive molecular
machines is devising any means to connect a reasonable collection of protein-sized
molecules into specific arrangements. We can already make (or harvest)
a wide variety of natural molecules (with known structures; perhaps slightly
modified) to serve as machine parts resembling "blocks, ropes, springs,
and semi-sticky surfaces" (as well as surfaces with catalytic activity),
which if arbitrarily joinable would be adequate to make a wide variety
of mechanical devices, including molecular computers and robots. It is
likely that such structures, combined with organic molecules like those
that can already be synthesized separately, could perform a wide variety
of synthetic organic reactions with selectivity due to positional control
(Drexler, 1981). Even though the first generation
of molecular mechanical devices would not be able to perform diamondoid
mechanosynthesis directly, it would lead to immense improvements in our
capabilities in the molecular realm, and would be on a direct path to full-fledged
nanotechnology.

One well-known scheme to join proteins together to form complex structures
(Drexler 1981, 1986, 1992)
is to learn to engineer folding polymers with many different specified
surface structures, and to use this ability to make "bricks" which self-assemble
in solution into a specific arrangement due to complementary surfaces.
The protein design issues to implement this scheme are very challenging;
there has been steady progress, but the level of success required for this
application still appears to be several years away. In contrast, the DGAP
method requires much less innovation to develop from already-demonstrated
technologies.

The DGAP invention is a method for assembling almost-natural molecular
building blocks (MBBs) into specific, almost-arbitrary arrangements, potentially
with hundreds of distinct block locations in each assembly. Blocks at different
locations in one assembly are allowed to be the same, which means that
a small library of distinct block types could suffice for many designs.
The assemblies of blocks could be joined recursively into larger structures.

MBBs could be joined in a fairly rigid fashion. Protein-based MBBs could
be held together in a compact fashion with covalent tethers at several
locations around the joined surfaces. Since the overall connectivity in
a network of MBBs could be essentially arbitrary (thus 3-D and polycyclic),
larger structures could be reasonably rigid as well. They are likely to
be able to be made rigid enough for positional control of chemical reactions,
though not strong enough, at least in the first generation, to apply the
significant forces needed for some uses of mechanosynthesis. (See Drexler
1992, pp.462-463, for a rigidity and strength analysis of a similar
system.) They will certainly be rigid enough for making "robot arms" which
could move other similar building blocks into contact with the correct
partners, and thus build similar devices more directly than by unassisted
self-assembly.

Besides rigid joints, rotating or tethered joints between MBBs are also
possible, either via different choices of chemistry for covalent bonding,
or by the use of building blocks which already incorporate such joints.

Since slightly modified natural proteins could be used as building blocks,
the resulting devices could incorporate biomolecules of interest in particular
applications, such as antibodies or enzymes.

The Basic Insight of DGAP

The basic idea, compared to the designed-complementary-surface approach,
is to try to remove or reduce the need to design molecular building blocks
with a variety of complementary interfaces by separating the two functions
of complementarity: (1) guidance of self-assembly and (2) a mechanically
strong connection of the MBBs.

Specifically, one could attach to each building block (at particular
sites on its surface, here called "C sites" for "complementary") a few
different single-stranded DNA molecules, and (at "P sites", for "permanent")
a few non-specific permanent binding sites. (The P sites will probably
be sites capable of forming covalent crosslinks, though other choices are
conceivable; several choices of chemistry for covalent crosslinking are
already in wide use in molecular biology. Also, it will sometimes be convenient
for an assembly consisting of a DNA strand and one or more P sites to be
added to each C site already on the protein, rather than for the protein
to start out with separate P sites and C sites.)

During assembly of several MBBs into one structure, the P sites are
prevented from reacting initially by maintaining a chemical environment
preventing reaction until the DNAs are hybridized, after which each P site
is only able to contact that P site with which it is meant to react. The
chemical environment is then changed to permit reaction (e.g. by the addition
of appropriate crosslinking reagents, active only at the P sites).

The problem of making a MBB by attaching a different DNA sequence to
each of several specific sites on a protein is the only part of the DGAP
invention without a direct precedent in current molecular biology. We have
developed several possible methods of solving it (the best of which we
are keeping under nondisclosure), and this is the main focus of our present
research; fully developing one of these methods and putting it into practice
will require some research and development in the laboratory. The kinds
of equipment and procedures required for performing the R&D on any
of our proposed DNA-attachment methods are standard in molecular biology.

For making large structures out of networks of linked MBBs, the average
MBB must be directly linked to more than two neighbors, preferably at least
3 or 4 if the structures should be rigid. The number of C sites and P sites
on each MBB needed to accomplish this depends on the specific design of
an assembly of MBBs; for ease of designing assemblies without this connectivity
being a limiting factor, we should be able to add different DNA sequences
to at least 6 different sites on one protein (taking advantage of the possibility
of connecting the C site to the middle of the DNA strand, so there would
be 12 different DNA sequences leaving the MBB). The methods we are investigating
should be able to accomplish this; if they can't, as few as 4 or even 3
separate C sites per protein would still be useful for many applications,
with more design effort required for some of them.

Major Issues not addressed in this document

Substantial issues affecting the feasibility of the DGAP invention and
affecting business plans based upon the DGAP invention have not been addressed
in this document. Additional information is available from Bruce Smith,
although some of this information will be governed by a non-disclosure
agreement. These issues include the following.

The specific attachment chemistry for both the C- and the P- sites remains
to be determined. Some possibilities for the attachment sites include using
surface cysteine residues (which are routinely introduced at new locations
via genetic engineering), or using the epsilon-amino group of lysine side
chains. One possible site-linkage chemistry is the creation of a disulfide
bond, which can be done with very high yield. The analysis to identify
a preferred chemistry has not been completed.

Choice of specific proteins to use as the core of the MBBs.

Methods for attaching a different specific DNA sequence to each distinct
attachment site on an MBB. As mentioned above, we know of several possible
methods, but are keeping their details under non-disclosure.

Near-term Applications would need to be more fully developed.
Some possibilities have been listed. A
choice will still be made regarding which application is easiest to
implement while having the highest early pay-off. The choice could be
influenced by the changing market opportunities that may exist two to
three years down the road.

Longer-term Applications would need to be more fully developed. Some possibilities
include

Analysis of related technology and approaches that may be competitive with
DGAP and which may affect the intellectual property position of the DGAP
invention. Examples include, but are not limited to, published papers and
abstracts using DNA complementarity to guide the assemblage of nanoscale
components, the use of DNA junctions to build nanoscale structures and
manipulators, and designing or evolving complementary surfaces to self-assemble.